PROJECT SUMMARY Infection of humans with Plasmodium falciparum parasites results in significant morbidity and mortality. In the face of drug-resistance, it is imperative to identify new antimalarial lead compounds to ensure the success of control and eradication efforts. Screening parasites in vitro against large numbers of small molecules has led to the identification of several antimalarials that are now at an advanced stage of development, with forward chemical genomic approaches being powerfully employed to identify putative molecular targets. We have developed conditional protein expression approaches that allow us to identify essential genes in the asexual cycle of Plasmodium parasites in red blood cells. In particular, we have recently identified the PfCRK4 gene as an essential regulator of DNA replication during asexual schizogony. Here, we propose to establish a new approach to identify small molecule inhibitors of essential genes, using parasite lines with conditional knockdown in specific target proteins. We will carry out small molecule inhibitor screens to identify compounds that synergize with PfCRK4 knockdown to inhibit parasite growth. We will perform drug screens with kinase inhibitor libraries biased to directly target PfCRK4, and structurally diverse bioactive libraries. We hypothesize that these screens will simultaneously reveal both specific inhibitors of the PfCRK4 enzyme, and secondary inhibitors that target molecules in pathways closely related to PfCRK4 that are synergistic with the PfCRK4-specific inhibitors. The specificity of the compounds will be further established by a combination of reverse chemical genetics and forward chemogenomic approaches. Of great value will be lead compounds against the essential putative drug target PfCRK4 that may inform drug development. We believe that the chemical genetic platform described here will simultaneously identify direct as well as secondary inhibitors of target pathways, important for the design of optimal drug combinations that might impede the emergence of drug-resistance.